Gamma-carboxyglutamic acid (Gla) is synthesized post-translationally in a vitamin K-dependent and CO2-requiring enzymatic reaction in the microsomal fraction of liver, kidney and bone tissue cells. Gla is a specialized calcium-binding amino acid which mediates a calcium and phospholipid dependent protelytic conversion in prothrombin to thrombin. Gla residues are found in a unique protein in bone named osteocalcin which has been characterized as a 6500 MW peptide containing 2 Gla residues/molecule. The function of osteocalcin in bone is unknown. This study proposes to determine its possible role in mineralization functioning either as a proenzyme, an inhibitor or a nucleating agent or a calcium transport protein. The microsomal osteocalcin substrate will be isolated and characterized to determine if osteocalcin-precursor is synthesized and undergoes vitamin K dependent carboxylation as the 6000 dalton peptide or as a higher molecular weight protein. To study the effect of vitamin K deficiency on bone mineralization processes, density-fractionation of ground bone will be performed to separate the least mineralized components from high density bone. A comparison of the distribution of fractions from densities of 1.4-2.1 g/cc in normal and vitamin K deficient bones will reveal if mineralization has been antagonized, either increased or decreased. In addition to these studies, clinical studies measuring urinary Gla excretion levels in patients with various metabolic disorders are being pursued. Both free and peptide bound Gla will be quantitated in 24 hour urines of patients with osteoporosis, Paget's disease of bone, osteomalacia and ectopic calcification disorders. The observed changes in urine Gla with a certain disorder may lend some insight into the functional nature of osteocalcin. Gla-protein(s) are also found associated with ectopic calcifications such as atherosclerotic plaque and scleroderma and may be responsible for the deposition of calcium. Their isolation and characterization is in progress.